Preparation of N-(phosphonoacetyl)-L-aspartic acid

ABSTRACT

Improved methods for preparation of the tetrasodium and disodium salts of N-(phosphonoacetyl)-L-aspartic acid (PALA) are disclosed. The present methods are well suited for preparation of these products in large amounts. A particular aspect of these methods includes a water-ethanol washing procedure for reducing impurities, including acetic acid, sodium acetate and ethanol, to an acceptable level.

The invention described herein was made in the course of work under agrant or award from the Department of Health, Education and Welfare.

BACKGROUND AND SUMMARY OF THE INVENTION

The present invention relates to improved methods for preparation ofN-(phosphonoacetyl)-L-aspartic acid, also known as PALA, in the form ofthe tetrasodium or disodium salt. More particularly, the presentinvention is concerned with a method for the large scale preparation ofthe tetrasodium salt and the disodium salt ofN-(phosphonoacetyl)-L-aspartic acid.

The compound, N-(phosphonoacetyl)-L-aspartic acid (PALA), was firstprepared as a rationally designed transition state analogue inhibitor ofaspartate transcarbamylase, as described by Stark et al., J. Biol.Chem., 246, 6599 (1971). Subsequent publications have presented animproved synthetic scheme for gram amounts of PALA, and havedemonstrated inhibition of pyrimidine nucleotide biosynthesis in vitro,as discussed by Stark et al., J. Biol. Chem., 249, 6945 (1974), as wellas in vivo, as discussed by Yoshida et al., J. Biol. Chem., 249, 6951(1974). PALA has been shown to be active against the B16, Lewis Lung,and P388 tumor systems, as indicated in the NCI screening program,Selected Agents List, Drug Evaluation Branch, DR and DP, Data throughAugust 31, 1976, p. 143.

While the synthesis of PALA is straightforward, the preparation of thetetrasodium salt or the disodium salt in kilogram quantities has provento be a major problem. The method of the present invention isparticularly well suited for the production of such quantities. Incopending U.S. Patent Application Ser. No. 851,382, filed Nov. 14, 1977,commonly assigned, there is described a method for the preparation ofthe tetrasodium and disodium salts of PALA, and this application isincorporated by reference herein.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The method for the preparation of the disodium salt of PALA inaccordance with the present invention is summarized in Outline I. As canbe seen in Outline I, the present method includes the initialpreparation of phosphonoacetyl chloride. Phosphonoacetyl chloride wasoriginally prepared in thionyl chloride at 60° C. The excess thionylchloride was removed by evaporation in vacuo, then the oily residue wasdissolved in dioxane. This solution was then added to dibenzyl aspartatein dioxane containing triethylamine at ˜15°. The insoluble triethylaminehydrochloride was filtered off, the dioxane was removed at reducedpressure, then the residue was dissolved in benzene. This was laterchanged to methylene chloride because of the OSHA restrictions on theuse of benzene. ##STR1## The organic solution was then washed severaltimes with water in order to remove unreacted acid chloride.

This procedure was acceptable on a small scale, however, many changeswere necessary in order to have a suitable production process.

Removal of the excess thionyl chloride in the preparation ofphosphonoacetyl chloride posed a major problem for scale-up. Thereaction was therefore attempted with one equivalent of thionyl chloridein dioxane. It was observed that, under completely anhydrous conditions,the reaction with dibenzyl aspartate gave ˜70-80% of an unknownmaterial. With the assumption that over chlorination was occurring, itseemed likely that, under the appropriate conditions, the P-Cl bondcould be hydrolyzed by the addition of water. This proved to be thecase. The chlorination has been successfully carried out in dioxaneusing from about 2.0 to 2.1 equivalents of thionyl chloride followed bythe addition of from about 1.0 to 1.2 equivalents of water. The dibenzylPALA obtained in this manner still contains a small amount of theunknown impurity. However, this can be eliminated through a saltformation or through formation of the cyclohexylammonium salt.

Other necessary modifications were made in order to improve the scale-upcapability of the process. Dioxane, because of its flammable and toxicproperties, was unattractive for large scale runs. A suitable substitutefor the chlorination reaction proved to be a mixture of glyme(1,2-dimethoxyethane) and methylene chloride in a volume ratio of 40:60.This solvent mixture is less toxic than dioxane and was determined to benonflammable.

The present method employs, as a key step in the sequence, the use ofdibenzyl aspartate. Reaction of the diester with phosphonoacetylchloride gives the water insoluble dibenzyl PALA. Preparation of thisintermediate allows for the convenient removal of unreacted, watersoluble acid chloride. Hydrolysis of dibenzyl PALA, purification of thereaction mixture by a batch ion exchange process, and adjusting anaqueous solution of the free acid to pH 9.2 gives the desiredtetrasodium salt.

The synthesized compound appeared to be free of significant organiccontamination as determined by NMR,O.R., and TLC, and was reported to beactive in the expected tumor systems. Analysis of the material, however,indicated the presence of as much as 13% of unknown inorganicimpurities. Because of the high water solubility of PALA, theseinorganic contaminants could not be removed by a simple water washprocedure.

In an effort to overcome this purity problem, an attempt was made topurify PALA by recrystallization. Glacial acetic acid was the firstsolvent chosen for this purpose, with the method being carried out asshown in Outline II. ##STR2##

Tetrasodium PALA dissolved in warm, glacial acetic acid but remained insolution on cooling. On the addition of ethanol, however, a white solidwas precipitated.

Elemental analysis of this material showed it to be the disodium salt.

Disodium PALA offers certain advantages over the tetrasodium compound,including the following:

(1) It is obtained directly in a solid form which eliminates thedifficult and time consuming trituration procedure necessary to solidifythe tetrasodium salt;

(2) It is less hygroscopic than the tetrasodium compound;

(3) It was determined that the compound could be prepared directly fromthe product mixture obtained on hydrolysis of dibenzyl PALA. Thisresulted in a shorter synthesis time and reduction in production costs.This also eliminated the ion exchange process shown in Outline I.

(4) The problem of over/or under titration associated with thepreparation of tetrasodium PALA was eliminated.

(5) Consistently low hydrogen analyses were obtained for the tetrasodiumsalt which was no longer a problem with the disodium compound;

(6) The synthesis was more adaptable to scale-up; and

(7) A good material balance was obtained for the disodium compound. Thisindicated that the unknown inorganic impurities were eliminated from theisolated product.

The general procedure for the preparation of disodium PALA is shown inOutline III. ##STR3##

Dibenzyl PALA was prepared from phosphonoacetyl chloride and dibenzylaspartate, then hydrolyzed with dilute, aqueous sodium hydroxide. Thecrude tetrasodium salt was then converted to disodium PALA by partialneutralization with glacial acetic acid. This basic scheme was used tosynthesize 25 kg. of the target compound and is currently being used forthe production of 60 kg. of the disodium salt.

In order to prepare 60 kilograms of disodium PALA, about 250 kilos ofdibenzyl aspartate must be made to obtain the target quantity. Thefollowing is an illustration of the steps employed and their applicationin going from a simple, classical esterification reaction tomultikilogram production.

Outline IV shows the esterification of aspartic acid.

This type of reaction is normally carried out in toluene or benzene withan acid catalyst. The equilibrium is shifted to give the ester bydistilling off the water azeotrope. The organic solvent is returned tothe reaction flask. This is the way the ester is formed for small scalework. However, to produce more than 500 pounds fast enough to servicethe needs of the present invention, major alterations must be made.##STR4##

In preparing dibenzyl aspartate, perchloroethylene was the solvent ofchoice. Its lack of flammability shortened the development time,appreciably, because of the ease in handling. This solvent forms anexcellent azeotrope with water. It boils high enough that thelarge-scale esterification is soon completed (within 3 hours) and, atthe same time, the product stability is not affected by thetemperature--at least during this short time of thermal contact.

A stoichiometric amount of p-toluenesulfonic acid was found to be mostadvantageous, and the optimum amount of benzyl alcohol was found to be4.5 moles per mole of aspartic acid. However, laboratory runsdemonstrated that when these components and the solvent were charged tothe reactor at room temperature, the mass would solidify. The problemwas solved by heating the solution of perchloroethylene, aspartic acidand benzyl alcohol to 65°-75° C. Then the p-toluenesulfonic acid wasadded. No insoluble salt formation occurred. In a similar fashion, afteresterification was completed, the reactor must be discharged at 80°-90°C.

Process control methods were devised based on thin layer chromatographicexamination of the reaction mixture. The optimum time for esterificationwas determined by TLC. Moreover, TLC markers of possible side-productswere obtained to relieve concern as to any tangential reactions. Forexample, the possible reaction of the toluenesulfonic acid with theamine of aspartic acid leading to the sulfonamide was shown not to occurunder the conditions of the desired esterification.

By retaining the product as the tosylate salt, the reactor could bedischarged directly into an optimum volume of acetone. The acetone wascooled with dry-ice. The pure ester precipitated immediately and onlyneeded to be washed with acetone before being dried. The product was ofsufficient quality that no impurities were carried into subsequent stepsin the production of PALA. Dibenzyl aspartate quality control includedthe established characteristics in IR, NMR, optical rotation, andelemental analyses.

The development of the present process is illustrated by the results ofdevelopment runs which are summarized in Table I.

                  Table I                                                         ______________________________________                                                   Amount                                                             Reactor Size                                                                             Aspartic Acid                                                                            Yield (%) Amount of Ester                               ______________________________________                                        250 ml.    13 g.      60-75     --                                            3 liter    133 g.     65-75     --                                            12 liter   532 g.     73        1,420 g.                                      76 liter   3,200 g.   64        7,400 g.                                       (20 gallon)                                                                  76 liter   3,200 g.   72        8,400 g.                                       (20 gallon)                                                                  380 liter  16,000 g.  64        37,500 g.                                      (100 gallon)                                                                 380 liter  16,000 g.  68        39,000 g.                                      (100 gallon)                                                                 ______________________________________                                    

Good yield retention has been obtained on scale-up. The purity is equalto or greater than 99% from the process with a present yield of about65%. The production per unit time is only limited by the size of thereactor. Moreover, the process information is developed to a level foreasy transfer to larger equipment.

The preparation of disodium PALA, as outlined in Outline III, results ina product contaminated with sodium aspartate, sodium acetate, unknownphosphorus--containing materials and the solvents acetic acid andethanol.

The dibenzyl PALA used for hydrolysis contains unreacted dibenzylaspartate which leads to sodium aspartate under the reaction conditions.The sodium acetate arises from the partial neutralization of thetetrasodium PALA and from the excess sodium hydroxide used in thehydrolysis.

Two impurities which require elimination are acetic acid and sodiumacetate. On a 500 g. scale, all of the sodium acetate and most of theacetic acid can be removed by extensive washing with ethanol. On alarger scale, this did not prove to be the case. The sodium acetatecould be removed, however, by a second precipitation from acetic acid.

On a 10 g. run, the ethanol and acetic acid were completely eliminatedby freeze-drying; however, this method was not operable when scaled upto 2 kg. This is an example of one of the vagaries of scale-uptechnology. This problem was solved by precipitating the crude disodiumPALA twice from water using ethanol. This method removed all of theacetic acid and sodium acetate. The second precipitation is a dropwiseaddition of an aqueous solution of PALA to the vortex of vigorouslystirred ethanol. The disodium salt precipitates as a granular solid.This method is suitable for quantities up to about 2 kg. For 25 kg. ofthe target material, this step alone would require almost 400 gallons ofethanol; for 60 kg., nearly 1000 gallons of ethanol would be needed.

It has now been determined that washing the PALA which is precipitatedfrom acetic acid with an appropriate water-ethanol solution cancompletely remove the acetic acid and sodium acetate. This purificationprocedure requires that 92±1% by volume aqueous ethanol be employed. Ifmore water is present, the material does not remain in solid form. Ifless water is used, the process is inoperative. The temperature at whichthis purification procedure is carried out is also critical. The optimumtemperature for conducting the washing procedure with the aqueousethanol is from about 26° to about 28° C. At temperatures above 30° C.,the compound turns from a solid to an oil. At temperatures below 25° C.,the procedure has been found to be ineffective. Thus the relativelynarrow limits of water concentration and temperature must be employed,otherwise the impurities cannot be removed from the product.

This practical leaching procedure reduces the volume of ethanol by atleast 50% and results in the saving of considerable man-hours. Anadditional problem of ethanol solvation had existed. Ethanolconcentration in the end product was at a 6-9% level. The leachingprocess causes a substitution of solvates, i.e., water for ethanol, thusreducing the ethanol to an acceptable 1 to 2%.

Thus there is provided a process that reduces acetic acid, sodiumacetate, and ethanol impurities to acceptable levels. Two majorcontaminants must still be controlled. These are aspartic acid and theunidentified phosphorus compounds.

The aspartic acid can be detected on TLC by spraying with ninhydrin.PALA is visualized on the chromatogram using a molybdate spray andappears as one spot in all of the solvent systems employed. However, anumber of phosphorus impurities, totaling 6% could be detected using P³¹NMR. It was believed that these phosphorus impurities did not originateduring either the hydrolysis or neutralization steps. As they were notpresent in the starting phosphonoacetic acid, they had to be formedduring the preparation of the dibenzyl PALA. This was confirmed by P³¹NMR which contained peaks other than that attributed to dibenzyl PALA.##STR5##

To prevent the formation of these impurities would require moreextensive research and possible routing alterations. The decision wasmade to minimize their formation, then remove the phosphoruscontamination from the dibenzyl PALA. This was carried out by the methodas shown in schematic in Outline V.

Upon preparation of the cyclohexylammonium salt of dibenzyl PALA, thedibenzyl PALA liberated from this purified salt was analyzed and foundto give 1 peak in the P³¹ NMR. In addition, both the liberated dibenzylPALA and the amine salt were subjected to the hydrolysis and partialneutralization steps. The P³¹ spectra of the resultant disodium PALAsamples confirmed that the phosphorus impurities could be eliminated bythis salt formation.

In addition to eliminating the phosphorus contaminants, the amine saltformation also removes the unreacted dibenzyl aspartate. This results ina final product free of aspartic acid contamination.

Scale-up development for the preparation of the pure salt has overcomesome potentially serious manipulative problems. The salt is prepared byadding from about 0.9 to about 1.0 equivalent of cyclohexylamine to anacetone solution of dibenzyl PALA. The product is insoluble in acetonewhereas a large percentage of the impurities remain in solution. It wasfound that the reaction must be run under anhydrous conditions withgentle mixing of the reactants, otherwise a gelatinous product willresult. On a multi-kilogram scale, this material would be nearlyimpossible to isolate. The purity of the product is upgraded to anacceptable level by recrystallization from absolute methanol.

Difficulties are encountered in the use of dioxane for the preparationof dibenzyl PALA. As mentioned, triethylamine hydrochloride is aninsoluble by-product of the reaction, and large volumes of solvent arerequired in order to maintain sufficient stirring. In addition, thereaction is exothermic, and the use of dioxane limits the extent ofcooling to ˜12°, which is the temperature at which dioxane freezes.

The solvent substituted for dioxane in this reaction was methylenechloride. This solvent offers the following advantages: (a) It isnonflammable; (b) It allows for a lower cooling temperature; (c) Thevolume of solvent is reduced in half; (d) The removal of triethylaminehydrochloride by filtration is eliminated since it is soluble in thereaction mixture; and (e) The evaporation of the solvent prior towork-up is no longer necessary.

The work-up involves aqueous washes of the methylene chloride solutionin order to remove unreacted acid chloride and triethylaminehydrochloride. A considerable emulsion problem was encountered duringthis washing procedure. This difficulty has been eliminated bysubstituting diluted hydrochloric acid for the water.

Additional process improvements include the fact that thecyclohexylammonium salt is hydrolyzed directly to the tetrasodium PALA.This eliminates the extra manipulation of releasing the dibenzyl PALAfrom the amine salt prior to hydrolysis.

Another improvement is the azeotropic removal of water from thehygroscopic dibenzyl PALA with chloroform prior to salt formation. Thisresults in a more crystalline salt and ultimately in a higher yield.

A final point is that the volume of water required for the hydrolysishas been reduced by 63% over that used in the initial synthetic work.This, of course, allows for larger scale runs to be made using the samesize equipment. At the bench scale, using as a maximum 50 l. flasks,this procedure has been used to prepare disodium PALA in ˜2 kg. lots.Incorporating all of the described modifications, a run using 50 and 100gallon Pfaudlers has been successfully carried out. At full scale, ˜15kg. of the target material can be produced per run using this sizeequipment. The process, as currently developed, is limited only by thesize of the equipment.

By the present method, the purity of the desired material has beenupgraded to a satisfactory IND level. This was accomplished by (1) aconvenient washing procedure which eliminates the acetic acid and sodiumacetate and reduces the ethanol content; and (2) by preparing thecyclohexylammonium salt of dibenzyl PALA which removes the aspartic acidand phosphorus-containing contaminants. In addition, the procedure hasbeen optimized for ease of scale-up, and the problems of processmanipulations have been solved.

Example 1 provides one sequence of steps in preparing the disodium saltof PALA while Example 2 provides a separate sequence of steps for thepreparation of the disodium salt of PALA.

EXAMPLE 1 Phosphonoacetyl chloride

A 50-gallon Pfaudler reactor was purged with nitrogen to a measuredoxygen content of <3%, then charged with methylene chloride (24 l.),1,2-dimethoxyethane (16 l.), N,N-dimethylformamide (1.76 l.), andphosphonoacetic acid (16.0 kg.; 114 moles). The stirred mixture wascooled to 5°, then thionyl chloride (28.56 kg.; 240.0 moles) was addedin a thin stream during 3 hours. The temperature of the reaction mixturewas maintained between 10° and 15° during the addition. The resultingsolution was heated at 30° for 3 hours, then the temperature wasincreased to 43° during the next hour. Additional methylene chloride(20.0 l.) was added, then the stirred solution was cooled (0°) andstored for 12 hours at 0°-3°. To this solution was added water (2.44 l.;137 moles), dissolved in 1,2-dimethoxyethane (8.0 l.), in a thin streamover a period of 4 hours while maintaining the reaction temperaturebetween 0° and 3°. After the addition was completed, the solution wasstirred an additional 0.5 hour at 0°, then immediately used in the nextreaction.

L-Aspartic acid, N-(phosphonoacetyl)-, dibenzyl ester

A 100-gallon Pfaudler reactor purged with nitrogen was charged insuccession with methylene chloride (120 l.), L-aspartic acid, dibenzylester p-toluenesulfonate (40.75 kg.; 83.93 moles), and triethylamine(58.6 l.). The stirred solution was cooled to 5°, then thephosphonoacetyl chloride (114.3 moles) was added in a thin stream during3 hours while the temperature was maintained between 10° and 18°.Additional triethylamine (6.0 l.) was added, and the mixture was stirredan additional 0.5 hour, then stored for 12 hours at room temperature.Methylene chloride (120 l.) was added, and the solution was washed insuccession with 10% aqueous hydrochloric acid (2×114 l.), 5% aqueoushydrochloric acid (3×114 l.) and water (114 l.). The organic layer wasdried over sodium sulfate (34.2 kg.) and magnesium sulfate (6.9 kg.)then concentrated in vacuo to 75 l. in a 100-gallon Pfaudler. Traceamounts of water were removed by co-distillation with chloroform (150 l.and 190 l.). Trace amounts of chloroform were removed by co-distillationwith acetone (150 l.). The resulting thick oil was diluted with acetone(300 l.), then used immediately in the next reaction.

L-Aspartic acid, N-(phosphonoacetyl)-, dibenzyl ester, complexed withcyclohexylamine

Cyclohexylamine (7478 g.; 75.54 moles) was added, in a thin stream, to agently stirred solution of L-aspartic acid, N-(phosphonoacetyl)-,dibenzyl ester (II) (36.50 kg.; 83.93 moles) in acetone (300 l.) at 20°during 20 minutes. The resulting mixture was stored for 12 hours at5°-10°, then the precipitate was collected and washed with acetone (70l.). The material was resuspended in acetone (230 l.), stirred for 30minutes, collected, washed with additional acetone (2×30 l.), then driedin vacuo (40°) to give 33.45 kg. (74.7%) of product. (33.2 kg.). Thismaterial was suspended in boiling methanol (527 l.). Celite (1.7 kg.)and cellulose (500 g.) were added, and the suspension was heated atreflux for 3 hours. Insolubles were removed by filtration (Celite). Theclear filtrate was diluted with acetone (400 l.), and the resultingmixture was stored for 16 hours at 5°-10°. The precipitated solid wascollected, washed with acetone (40 l.), then dried in vacuo (40°) togive 17.49 kg. (52.6% recovery) of purified product. The mother liquorwas concentrated in vacuo, and an additional 7.31 kg. of material wasobtained to give a total of 24.80 kg. (75% recovery) of purifiedproduct.

L-Aspartic acid, N-(phosphonoacetyl)-, tetrasodium salt

To a cold (2°), stirred solution of sodium hydroixde (97%) (7532 kg.;182.7 moles) in distilled water (114 l.) was added L-aspartic acid,N-(phosphonoacetyl)-, dibenzyl ester, complexed with cyclohexylamine(23.8 kg.; 44.5 moles) during 30 minutes while maintaining thetemperature of the mixture at 10°-15°. The mixture was stirred at 15°for 4 hours then stored at 10° for 16 hours. The aqueous solution waswashed with methylene chloride (4×75 l.). Decolorizing carbon (700 g.)and Celite (1 kg.) were added to the aqueous solution. The mixture wasstirred for 30 minutes then clarified by filtration. The filtrate wasdiluted with ethanol (410 l.) then stored at 10° for 8 hours. Thesupernatant alcohol layer was removed, and the heavy oil washed withadditional ethanol (6.5 l.) then immediately used in the next reaction.

L-Aspartic acid, N-(phosphonoacetyl)-, disodium salt .1.3 H₂ O.0.13 EtOH

Glacial acetic acid (70.0 l.) was added to the L-aspartic acid,N-(phosphonoacetyl)-, tetrasodium salt oil from the above reaction. Themixture was stirred at room temperature for 30 minutes, clarified byfiltration, then ethanol (238 l.) was added to the stirred solutionduring 1.75 hours. The mixture was stirred an additional hour thenstored at 10° for 20 hours. The resulting precipitate was collected thenwashed with ethanol (12.0 l.). This material was washed by resuspensionwith 92% ethanol - 8% water (60 gal.) at 27° for 15 hours in a100-gallon Pfaudler reactor. The solid was collected then washed on thefilter with absolute ethanol (4×1.5 gal.). The solid was washed a secondtime with 92% ethanol - 8% water (60 gal.) at 26° for 15 hours. Thesolid was collected, washed on the filter with absolute ethanol (5×2gal.), then dried to constant weight in vacuo (first at roomtemperature, then at 40°) to give 12,154 g. (76%) of purified product.The melting point of this material is non-determinant.

    ______________________________________                                        Anal.                                                                         Calc'd, for C.sub.6 H.sub.7.7 NO.sub.8 P . 2.3 Na . 1.3 H.sub.2 O . 0.13      EtOH                                                                          C            H        N        P      Na                                      ______________________________________                                                22.44    3.33     4.18   9.24   15.78                                 Found   22.69    3.43     4.20   9.27   15.81                                 ______________________________________                                    

Sodium analysis indicates a composition of

70% di-NaPALA

30% tri-NaPALA

Based on empirical formula and spectral data,

% H₂ O=7.0%

% etOH=1.8%

Spectral Data

Infrared (Nujol): Major bands: 3400-3200, 2920, 2850, 1730-1690,1650-1580, 1460, 1370, 1170-1140, 1070-1030, 910-870 cm⁻¹

Nuclear Magnetic Resonance (D₂ O): δ 4.70 (HOD); 4.50 (t, 1, --CH--,J=6.0 Hz); 3.60 (q, 0.3, --CH₂ -- of ethanol, J=7.0 Hz); 3.00-2.50 (m,4, methylene H); 1.15 (t, 0.4, --CH₃ of ethanol, J=7.0 Hz)

    ______________________________________                                        Optical Rotation:                                                             Observed         Literature                                                   ______________________________________                                        [α].sup.23.sub.D + 14.56° (c, 0.206                                               [α].sup.22.sub.D + 14.86° (c, 1.998             in H.sub.2 O)    in H.sub.2 0)                                                Chromatography:                                                               Thin Layer Chromatography                                                     (Cellulose, Quanta/Gram Q2F Glass Plates)                                           Solvent System        R.sub.f Value                                     ______________________________________                                        1.   Lithium chloride (0.6 M)-                                                     ethanol-ammonium hydroxide                                                    (5:5:1)                0.47                                              2.   Ethanol-water (2:3)    0.78                                              3.   Ethanol-ammonium hydroxide-                                                                          0.22                                                   water (6:1:3)          (elongated)                                       4.   n-Butanol-acetic acid-water                                                                          0.30                                                   (5:2:3)                (tailing)                                         ______________________________________                                         Detection: (a) Ninhydrin (b) Phospray                                         Quantity Spotted: 300 μg.                                                  Results: The compound moves as one phospray positive spot in each of the      solvent systems. No aspartic acid was observed on spraying with ninhydrin                                                                              

EXAMPLE 2 Phosphonoacetyl chloride

To a stirred mixture of phosphonoacetic acid (2000 g.; 14.28 moles),N,N-dimethylformamide (208.8 g.; 2.856 moles), and dioxane (7.15 l.) wasadded, dropwise, thionyl chloride (3568 g.; 29.99 moles) during 1.5hours. The temperature was maintained below 30° during the addition. Theresulting solution was heated at 45° for 2.5 hours then cooled to 5°.Water (283 ml.; 15.7 moles) dissolved in dioxane (2.5 l.) was thenadded, dropwise, over a period of 2 hours. The temperature was keptbelow 10° during the addition. This solution of acid chloride wasstirred at 5°-10° for 40 minutes then used in the following reactionwithout further characterization. A second chlorination was carried outconcurrently, under the same conditions, using identical quantities ofreactants.

L-Aspartic acid, N-(phosphonoacetyl)-, dibenzyl ester

A stirred suspension of L-aspartic acid, dibenzyl esterp-toluenesulfonate (4625 g.; 9.525 moles) in dioxane (20.0 l.) wascooled to 15°, then triethylamine (4820 g.; 47.63 moles) was added, in athin stream, during 1 hour. The resulting solution was stirred for 20minutes, then the above solution of phosphonoacetyl chloride, preparedfrom 14.28 moles of the corresponding acid, was added, dropwise, over aperiod of 5 hours. The temperature was maintained below 20° during theaddition. Additional triethylamine (1162 g.; 11.48 moles) was added andthe reaction mixture was stirred for 1 hour. After standing for 8 hoursat room temperature, the mixture was diluted with acetone (5.5 l.),stirred for 15 minutes, then the insolubles were collected on a filterand washed with dioxane (10.0 l.). A second reaction was carried outconcurrently, under the same conditions, using identical amounts ofmaterials. The filtrates from the two runs were combined andspin-evaporated in vacuo. The residue (orange, viscous oil) wasdissolved in methylene chloride (110.0 l.), then the organic solutionwas gently washed with water (6×30.0 l.). After drying the solution oversodium sulfate (11.3 kg.) and magnesium sulfate (2.3 kg.), theinsolubles were filtered off (Celite pad), and the filtrate wasevaporated in vacuo to constant weight; yield of dibenzyl PALA, 7970 g.(96.1%). This yellow, viscous oil was suitable for furthertransformation.

L-Aspartic acid, N-(phosphonoacetyl)-, dibenzyl ester, complexed withcyclohexylamine

Cyclohexylamine (1815 g.; 18.30 moles) was added, dropwise, to a cold(7°), stirred solution of L-aspartic acid N-(phosphonoacetyl)-, dibenzylester (7970 g.; 18.30 moles) in acetone (24.0 l.) during 1.25 hours. Thetemperature was maintained below 15° during the addition. The coolingbath was removed, and the resulting mixture was stirred for 1 hour. Themixture was stored at room temperature for 6 hours, then theprecipitated solid was collected on a filter, washed with acetone (15.0l), and dried; yield, 4932 g.; m.p., 176.5°-177.5°. This material wasrecrystallized from boiling methanol (35.0 l.) then dried to give 1663g. of the purified salt; m.p., 178°-181°; literature m.p., 186°-188°.The mother liquor was concentrated in vacuo to a volume of 20.0 l. Thesolution was diluted with acetone (16.0 l.) and cooled (-10°) to give anadditional 967 g. of product; m.p., 177° -180°. A third crop of material(429 g.) was obtained by evaporating the above methanol-acetone filtrateto near dryness and suspending the residue in acetone (5.0 l.); totalamount of the purified amine salt suitable for further transformation,3059 g. (62.0% recovery).

L-Aspartic acid, N-(phosphonoacetyl)-, tetrasodium salt

To a cold (5°), stirred solution of sodium hyroxide (1291 g.; 32.28moles) in water (20.5 l.) was added, in portions, during 30 minutes,L-aspartic acid, N-(phosphonoacetyl)-, dibenzyl ester, complexed withcyclohexylamine (3059 g.; 5.378 moles if the amine salt has the sameempirical formula as the analytically pure sample). The reaction mixturewas stirred at 5°-15° for 3.5 hours, then extracted with methylenechloride (2×8.5 l.) and ether (1×8.5 l.). The aqueous solution wasclarified by filtration, concentrated in vacuo (<35°; 3-5 mm. Hg) to avolume of 14.6 l., then diluted with ethanol (51.4 l.). The resultingmixture was stirred for 1 hour and stored at room temperature for 12hours. The aqueous ethanol solution was removed giving crude product asa light yellow oil suitable for further transformation.

L-Aspartic acid, N-(phosphonoacetyl)-, disodium salt

Glacial acetic acid (8.0 l.) was added to the above precipitated oil[crude L-aspartic acid, N-(phosphonoacetyl)-, tetrasodium salt preparedfrom 3059 g. of the amine salt]. The mixture was stirred at roomtemperature for 30 minutes, then a gelatinous insoluble was filteredoff. The clear, light yellow filtrate was diluted with ethanol (24.0l.). The resulting mixture was stirred for 1.75 hours, then theprecipitated material was collected on a filter. The solid was suspendedin ethanol (14.5 l.), and the mixture was vigorously stirred for 1 hour.The product was collected on four filters then partially dried byspin-evaporation in vacuo (30°-45°; aspirator pressure than 3-5 mm. Hg).The lumpy material (2870 g.) was dissolved in water (5.25 l.), thesolution was clarified by filtration, then the filtrate (˜6.9 l. volume)was diluted with ethanol (21.0 l.). The resulting mixture was stirredfor 30 minutes, then the precipitated oil was allowed to settle (1hour). The aqueous ethanol solution was removed, and the oil was washedonce with ethanol (4.3 l.). This material was dissolved in water (8.15l), and the solution (9.8 l.) was divided into three portions (two of4.0 l.; one of 1.8 l.). Each portion was added, during 13 hours, to thevortex of vigorously stirred ethanol (10×aqueous volume: 2×40.0 l.;1×18.0 l.). After stirring the mixtures for 2 hours, the water-ethanolsolutions were siphoned off, and the solid from the three precipitationswas combined. The material was stirred for 30 minutes in ethanol (10.0l.), collected on a filter, then dried to constant weight in vacuo atroom temperature over phosphorus pentoxide. The dried product (1748.0g.) was passed through a 150μ, stainless steel sieve and thoroughlyblended to give (V) as a white powder.

    ______________________________________                                        Anal.                                                                         Calc'd. for C.sub.6 H.sub.7.6 NO.sub.8 P . 2.4 Na . 2 H.sub.2 O . 0.5         C.sub.2 H.sub.6 O                                                             C            H        N        P      Na                                      ______________________________________                                                22.91    4.01     3.82   8.44   15.04                                 Found   23.16    3.76     3.79   8.57   15.18                                 ______________________________________                                    

Sodium analysis indicates a composition of

60% di-Na PALA

40% tri-Na PALA

Based on the empirical formula,

% H₂ O=9.8%

% etOH=6.3%

Spectral Data: Nuclear Magnetic Resonance (D₂ O): δ 1.17 (t, 1.5, --CH₂of ethanol); 2.74 (d, 2, --CH₂ α to --CH); 2.77 (d, 2, J=20 Hz, --CH₂ αto P); 3.63 (q, 1, --CH₂ of ethanol); 4.48 (t, 1, --CH)

    ______________________________________                                        Optical Rotation:                                                             Observed         Literature                                                   ______________________________________                                        [α].sup.22.5.sub.D + 14.73 (c,                                                           [α].sup.22.sub.D + 14.86 (c,                           2.098 in water)  1.998 in water)                                              Chromatography:                                                               Thin Layer Chromatography                                                     (Cellulose, Quanta/Gram Q2F Glass Plates)                                           Solvent System        R.sub.f Value                                     ______________________________________                                        1.   Lithium chloride (0.6 M)-                                                     ethanol-ammonium hydroxide                                                    (5:5:1)                0.52                                              2.   Ethanol-water (2:3)    0.72                                              3.   Ethanol-ammonium hydroxide-                                                                          0.16                                                   water (6:1:3)          (elongated)                                       4.   n-Butanol-acetic acid-water                                                                          0.22                                                   (5:2:3)                (tailing)                                         ______________________________________                                         Detection: (a) Ninhydrin (b) Phospray                                         Results: The compound moves as one phospray positive spot in each of the      solvent systems. No aspartic acid was observed on spraying with ninhydrin                                                                              

It is thought that the invention and many of its attendant advantageswill be understood from the foregoing description, and it will beapparent that various changes may be made in the methods as describedherein without departing from the spirit and scope of the invention orsacrificing its material advantages, the forms hereinbefore describedbeing merely preferred embodiments thereof.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. In a method for thepreparation of a sodium salt of N-(phosphonoacetyl)-L-aspartic acid byreacting L-aspartic acid, dibenzyl ester p-toluenesulfonate withtriethylamine, followed by the addition of phosphonoacetyl chloride toproduce the N-(phosphonoacetyl)-L-aspartic acid moiety in the form ofthe dibenzyl ester, wherein the carboxyl groups of theN-(phosphonoacetyl)-L-aspartic acid are esterified, addingcyclohexylamine under anhydrous conditions to produce thecyclohexylamine salt of said dibenzyl ester, reacting saidcyclohexylamine salt with sodium hydroxide to produce L-aspartic acid,N-(phosphonoacetyl)-, tetrasodium salt, and reacting said tetrasodiumsalt with acetic acid to produce L-aspartic acid, N-(phosphonoacetyl)-,disodium salt, the improvement which comprises subjecting the obtaineddisodium salt to a water-ethanol washing procedure, employing 92±1% byvolume aqueous ethanol, said washing procedure being carried out at atemperature of from about 26° to about 28° C., thereby reducingimpurities, including acetic acid, sodium acetate and ethanol, to anacceptable level.